Lipoxygenase

ABSTRACT

The invention provides sequence information of a microbial protein having lipoxy-genase activity and a method of producing the protein by recombinant DNA technology. More specifically, the inventors have isolated a gene encoding a lipoxygenase from  Gaeu - mannomyces graminis , cloned it into an  E. coli  strain and sequenced it. A comparison shows less than 25% identity to known lipoxygenase sequences, the closest being human 15S li-poxygenase. The inventors have expressed the lipoxygenase recombinantly and found that the recombinant lipoxygenase is glycosylated.

FIELD OF THE INVENTION

[0001] The present invention relates to a polynucleotide encoding alipoxygenase and its use for recombinant production of a lipoxygenase.The invention also relates to a method of obtaining a lipoxygenase byscreening a DNA library with specific probes.

BACKGROUND OF THE INVENTION

[0002] Lipoxygenase is an enzyme that catalyzes the oxygenation oflinoleic acid and produces a hydroperoxide. It is classified in EnzymeNomenclature as EC 1.13.11.12. The enzyme is widely distributed inplants and animals. Encoding genes have been isolated from varioussources, and the sequences have been published. Thus, GENESEQP W93832and Genbank U78294 give the sequence of human 15S lipoxygenase.

[0003] Microbial lipoxygenases are known from a yeast Saccharomycescerevisiae, a thermophilic actinomycete Thermoactinomyces vulgaris, fromfungus Fusarium oxysporum, Fusarium proliferatum and Gaeumannomycesgraminis (Su and Oliw, J. Biological Chemistry, 273 (21), 13072-13079(1998)). No isolated gene encoding a microbial lipoxygenase has beendescribed.

[0004] The prior art describes various uses of lipoxygenase, e.g. as afood additive to bread dough or noodles.

SUMMARY OF THE INVENTION

[0005] Here we for the first time provide sequence information of amicrobial protein having lipoxygenase activity and a method of producingthe protein in industrial scale. More specifically, the inventors haveisolated a gene encoding a lipoxygenase from Gaeumannomyces graminis,cloned it into an E. coli strain and sequenced it. The genome of G.graminis contains approximately 60% of the G and C nucleotides, whichmade this work very difficult. A comparison shows less than 25% identityto known lipoxygenase sequences, the closest being human 15Slipoxygenase. The inventors have expressed the lipoxygenaserecombinantly.

[0006] Accordingly, the invention provides a polypeptide havinglipoxygenase enzyme activity which:

[0007] a) has an amino acid sequence which has at least 50% identitywith the mature polypeptide of SEQ ID NO: 2 or 23;

[0008] b) is encoded by a nucleic acid sequence which hybridizes at 55°C. with a complementary strand of the nucleic acid sequence encoding themature polypeptide of SEQ ID NO: 1 or a subsequence thereof having atleast 100 nucleotides;

[0009] c) has an amino acid sequence which can be obtained from themature poly-peptide of SEQ ID NO: 2 or 23 by substitution, deletion,and/or insertion of one or more amino acids; or

[0010] d) is encoded by the lipoxygenase-encoding part of the DNAsequence cloned into a plasmid present in Escherichia coli depositnumber DSM 13586.

[0011] The invention also provides a polynucleotide which comprises:

[0012] a) the partial DNA sequence encoding a mature lipoxygenase clonedinto a plasmid present in Escherichia coli DSM 13586,

[0013] b) the partial DNA sequence encoding a mature lipoxygenase shownin SEQ ID NO: 2 or 23,

[0014] c) an analogue of the sequence defined in a) or b) which encodesa lipoxygenase and

[0015] i) has at least 50% identity with said DNA sequence, or

[0016] ii) hybridizes at low stringency with a complementary strand ofsaid DNA sequence or a subsequence thereof having at least 100nucleotides,

[0017] iii) is an allelic variant thereof, or

[0018] d) a complementary strand of a), b) or c).

[0019] Other aspects of the invention provide a nucleic acid constructcomprising the polynucleotide, a recombinant expression vectorcomprising the nucleic acid construct, and a recombinant host celltransformed with the nucleic acid construct. The invention also providesa recombinant method of producing the lipoxygenase, an oligonucleotideprobe based on SEQ ID NO: 2 or 23 and a method of obtaining alipoxygenase by screening a eukaryotic DNA library using the probe basedon SEQ ID NO: 2.

[0020] Further, the invention provides a dough composition comprising amanganese lipoxygenase and a method for preparing a dough or a bakedproduct made from dough, comprising adding a manganese lipoxygenase tothe dough. The invention also provides a method of oxygenating asubstrate selected from the group consisting of linolenic acid,arachidonic acid, linoleyl alcohol and a linoleic acid ester comprisingcontacting the substrate in the presence of oxygen with a manganeselipoxygenase. Finally, the invention provides a detergent compositioncomprising a manganese lipoxygenase and a surfactant.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Genomic DNA Source

[0022] DNA encoding the lipoxygenase (LOX) may be derived from fungi,particularly Ascomycota, more particularly Ascomycota incertae sedise.g. Magnaporthaceae, such as Gaeumannomyces, or anamorphicMagnaporthaceae such as Pyricularia, or alternatively anamorphicAscomycota such as Geotrichum. An example is G. graminis, e.g. G.graminis var. graminis, G. graminis var. avenae or G. graminisvar.tritici, particularly the strain G. graminis var. graminis CBS903.73, G. graminis var. avenae CBS 870.73 or G. graminis var.triticiCBS 905.73. The CBS strains are commercially available fromCentraalbureau voor Schimmelcultures, Baarn, the Netherlands.

[0023] The inventors obtained two LOX-encoding DNA sequences fromstrains of Gaeumannomyces graminis and found that they have thesequences shown in SEQ ID NO: 1 and 22. They inserted a LOX-encodinggene into a strain of Escherichia coli and deposited it as E. coli DSM13586 on Jul. 5, 2000 under the terms of the Budapest Treaty with theDSMZ—Deutsche Sammlung von Microorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany. The deposit wasmade by Novo Nordisk A/S and was later assigned to Novozymes A/S.

[0024] Lipoxygenase

[0025] The lipoxygenase of the invention is a manganese lipoxygenase,i.e. it has lipoxygenase activity (EC 1.13.11.12) with manganese in theprosthetic group. It is glycosylated and may have a molecular weight inthe range 90-110 kDa, particularly 95-105 kDa. It is thermostable with atemperature optimum of 65-90° C., particularly 75-85° C. Thelipoxygenase is stable against LAS (linear alkyl-benzene sulfonate) upto 400 ppm at pH 10. Mn-Lipoxygenase is enzymatically active between pH5-12 with a broad optimum at pH 6-8.

[0026] A recombinant lipoxygenase may have a higher glycosylation and ahigher thermostability. The recombinant lipoxygenase may have amolecular weight in the range 90-110 kDa, particularly 95-105 kDa. Itmay have a temperature optimum of 65-90° C., particularly 75-85° C.

[0027] Recombinant Expression Vector

[0028] The expression vector of the invention typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a selectable marker, atranscription terminator, a repressor gene or various activator genes.The vector may be an autonomously replicating vector, or it may beintegrated into the host cell genome.

[0029] Production by Cultivation of Transformant

[0030] The lipoxygenase of the invention may be produced by transforminga suitable host cell with a DNA sequence encoding the lipoxygenase,cultivating the transformed organism under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture.

[0031] The host organism may be a eukaryotic cell, in particular afungal cell, such as a yeast cell or a filamentous fungal cell, e.g. astrain of Aspergillus, Fusarium, Trichoderma or Saccharomyces,particularly A. niger, A. oryzae, F. graminearum, F. sambucinum, F.cerealis or S. cerevisiae. The production of the lipoxygenase in suchhost organisms may be done by the general methods described in EP238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

[0032] Nucleotide Probe

[0033] A nucleotide probe may be designed on the basis of the DNAsequence of SEQ ID NO: 1 or the polypeptide sequence of SEQ ID NO: 2,particularly the mature peptide part. The probe may be used in screeningfor LOX-encoding DNA as described below.

[0034] A synthetic oligonucleotide primer may be prepared by standardtechniques (e,g, as described in Sambrook J, Fritsch E F, Maniatis T(1989) Molecular cloning: a laboratory manual (2^(nd) edn.) Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) on the basis of the maturepart of the amino acid sequence in SEQ ID NO: 2 or the correspondingpart of the DNA sequence. It may be a degenerate probe and willtypically contain at least 20 nucleotides.

[0035] Screening of Eukaryotic DNA Library

[0036] A polypeptide with lipoxygenase activity may be obtained by amethod comprising:

[0037] a) preparing a eukaryotic DNA library,

[0038] b) screening the library to select DNA molecules which hybridizeto the probe described above,

[0039] c) transforming host cells with the selected DNA molecules,

[0040] d) cultivating the transformed host cells to express polypeptidesencoded by the DNA molecules, and

[0041] e) assaying the expressed polypeptides to select polypeptideshaving lipoxygenase activity.

[0042] The eukaryotic DNA library may be prepared by conventionalmethods. It may include genomic DNA or double-stranded cDNA derived fromsuitable sources such as those described above.

[0043] Molecular screening for DNA sequences may be carried out bypolymerase chain reaction (PCR) followed by hybridization.

[0044] In accordance with well-known procedures, the PCR fragmentgenerated in the molecular screening may be isolated and subcloned intoa suitable vector. The PCR fragment may be used for screening DNAlibraries by e.g. colony or plaque hybridization.

[0045] Hybridization

[0046] The hybridization is used to indicate that a given DNA sequenceis analogous to a nucleotide probe corresponding to a DNA sequence ofthe invention. The hybridization may be done at low, medium or highstringency. One example of hybridization conditions is described indetail below.

[0047] Suitable conditions for determining hybridization between anucleotide probe and a homologous DNA or RNA sequence involvespresoaking of the filter containing the DNA fragments or RNA in 5×SSC(standard saline citrate) for 10 min, and prehybridization of the filterin a solution of 5×SSC (Sambrook et al. 1989), 5× Denhardt's solution(Sambrook et al. 1989), 0.5% SDS and 100/g/ml of denatured sonicatedsalmon sperm DNA (Sambrook et al. 1989), followed by hybridization inthe same solution containing a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity>1×10⁹ cpm/μg) probe for 12 hours at approx. 45□° C.The filter is then washed two times for 30 minutes in 2×SSC, 0.5% SDS ata temperature of at least 55□ C., particularly at least 60□ C., moreparticularly at least 65□ C., e.g. at least 70□ C., or at least 75□ C.

[0048] Molecules to which the oligonucleotide probe hybridizes underthese conditions are detected using an x-ray film.

[0049] Alignment and Identity

[0050] The nucleotide sequence of the invention may have an identity tothe disclosed sequence of at least 75% or at least 85%, particularly atleast 90% or at least 95%, e.g. at least 98%.

[0051] For purposes of the present invention, alignments of sequencesand calculation of identity scores were done using a Needleman-Wunschalignment (i.e. global alignment), useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R.Pearson and D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA”, Methods inEnzymology, 183:63-98).

[0052] Use of Lipoxygenase

[0053] A manganese lipoxygenase such as that described above may be usedin the following application, e.g. in analogy with the indicatedpublications.

[0054] The lipoxygenase can be used as an additive to dough for bakedproducts such as bread, biscuits and cakes. Thus, the lipoxygenase canbe used in a process for making bread, comprising adding thelipoxygenase to a dough, kneading the dough and baking the dough to makethe baked product. SU 426640 A, JP 58190346 A[SLK1], JP 1165332 A[SLK2],JP 8322456,[SLK3] JP 10028516[SLK4], JP 08322456, JP 2964215. It canalso be used in the preparation of noodles as described in JP 11299440A.

[0055] The lipoxygenase may be used for bleaching, e.g. bleaching ofbeta-carotene, wheat flour or wheat dough. U.S. Pat. Nos.1,957,333-1,957,337.

[0056] It can also be used for oxidizing mixtures of fatty acids tohydroperoxy fatty acids, as accelerators of lipid peroxidition, and asanalytic tools to estimate linoleic and linolenic acids contents ofcertain oils.

[0057] The invention provides a detergent composition comprising thelipoxygenase and a surfactant, particularly an anionic surfactant suchas LAS (linear alkyl-benzene sulfonate). Advantageously, thelipoxygenase has good stability in the presence of such surfactants. Thedetergent may be formulated as described in U.S. Pat. No. 3,635,828[SLK5]or U.S. Pat. No. 5,789,362[SLK6]. The lipoxygenase can also beused to bleach stains from fabrics or hard surfaces as described in DK9800352[SLK7]. Advantageously, The lipoxygenase can be used formodification of starch as mentioned in JP 09163953, EP772980, JP2000-106832. Also it can be used for protein modification as describedin EP 947142, DE 19840069 or JP 61078361, or modification of oil(production of conjugated fatty acid) as mentioned in JP 5905128, U.S.Pat. No. 3,729,379.

[0058] The lipoxygenase can be used for cross-linking a protein byoxidases, such as laccase, bilirubin oxidase etc. EP 947142.

[0059] The lipoxygenase can be used to obtain improved glutinousness andimproved flavor of marine paste product such as Kamaboko, Hanpen, byadding lipoxygenase to fish meat. JP 61078361.

[0060] The lipoxygenase can be used to produce a process tomato product.It can be used for tomato pasta, salsa, ketchup and so on. EP 983725.

[0061] The lipoxygenase can be used for production of hydroperoxy fattyacid by reacting lipoxygenase with unsaturated 4-24C fatty acid. JP11029410.

[0062] The hydroperoxides of linoleic acid or linolenic acid can beconverted further to e.g. growth regulatory hormone jasmonic acid, andthe product from arachidonic acid can be converted to physiologicaleffectors leukotrienes and lipoxins.

[0063] Application of lipoxygenase should not be limited to the examplesmentioned above. Since hydroperoxide, the product of lipoxygenasereaction, is good oxidant to create radical, lipoxygenase can be usedfor any other applications utilizing oxidation reaction, such asbleaching of food material or textile dyes, or polymerization ofchemical compounds to produce plastic material or fiber.

[0064] Assay for Lipoxygenase Activity

[0065] The lipoxygenase activity was determined spectrophotometricallyat 25° C. by monitoring the formation of hydroperoxides. For thestandard analysis, 10 μL enzyme was added to a 1 mL quartz cuvettecontaining 980 μL 25 mM phosphate buffer (pH 7.0) and 10 μL of substratesolution (10 mM linolenic acid dispersed with 0.2%(v/v) Tween20). Theenzyme was typically diluted sufficiently to ensure a turn-over ofmaximally 10% of the added substrate within the first minute. Theabsorbance at 234 nm was followed and the rate was estimated from thelinear part of the curve. One unit causes an increase in absorbance at234 nm of 0.001/min.

[0066] Determination of Substrate Specificity

[0067] The substrate specificity of the lipoxygenase was studied usingthe standard assays condition with a number of different compounds assubstrate. All substrates were produced as dispersions with 0.2%(v/v)Tween20. The amount of compound added to make up these stock solutionswas determined by mass, since viscosity made accurate measurement ofvolume impossible. The limiting rate constant and the specificityconstant were determined by varying the amount of substrate added in theassays. The resulting rates were plotted against the concentration ofsubstrate used. Finally, the plots were fitted by non-linear leastsquares regression to the theoretical hyperbolic curve of theMichaelis-Menten equation. The cis-trans-conjugated hydro(pero)xy fattyacids were assumed to have a molecular extinction coefficient of 23,000M⁻¹ cm⁻¹.

EXAMPLES

[0068] Materials and Methods

[0069] Molecular cloning techniques are described in Sambrook et al.(1989).

[0070] The following commercial plasmids and E. coil strains were usedfor sub-cloning and DNA library construction:

[0071] pT7Blue (Novagen)

[0072] pUC19 (TOYOBO, Japan)

[0073]E. coli JM109 (TOYOBO, Japan)

[0074]E. coli DH12□ (GIBCO BRL, Life Technologies, USA)

[0075] The following commercial Kits were used for cDNA cloning;

[0076] cDNA Synthesis Kit (Takara, Japan)

[0077] Marathon cDNA Amplification Kit (Clontech, USA)

[0078] Oligo dT cellulose powder (Invitrogen, Netherlands)

[0079] Labeling and detection of hybridization probe was done usingDIG-labeling and detection Kit (Boehringer Manheim). Nylon membraneHybond-N+ (Amersham, England) was used for DNA transfer for bothsouthern blotting and colony hybridization.

[0080] Media and Buffer Solution

[0081] COVE-ar: per liter 342.3 g sucrose, 20 ml COVE salt solution, 10mM acrylamide, 15 mM CSCl₂, 30 g Agar noble (Difco)

[0082] COVE2-ar: per liter 30 g sucrose, 20 ml COVE salt solution, 10 mMacrylamide, 30 g Agar noble (Difco)

[0083] COVE salt solution: per liter 26 g KCl, 26 g MgSO₄-7H₂O, 76 gKH₂PO₄, 50 ml Cove trace metals.

[0084] Cove trace metals: per liter 0.04 g NaB₄O₇-10H₂O, 0.4 gCuSO₄-5H₂O, 1.2 g FeSO₄-7H₂O, 0.7 g MnSO₄-H₂O, 0.7 g Na₂MoO₂-2H₂O, 0.7 gZnSO₄-7HpO.

[0085] AMG trace metals: per liter 14.3 g ZnSO₄-7H₂O, 2.5 g CuSO₄-5H₂O,0.5 g NiCl₂, 13.8 g FeSO₄, 8.5 g MnSO₄, 3.0 g citric acid.

[0086] YPG: per liter 4 g yeast extract, 1 g KH₂PO₄, 0.5 g MgSO₄-7H₂O,15 g glucose, pH 6.0.

[0087] STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl₂.

[0088] STPC: 40% PEG4000 in STC buffer.

[0089] Cove top agarose: per liter 342.3 g sucrose, 20 ml COVE saltsolution, 10 mM Acelamide, 10 g low melt agarose.

[0090] MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0.

[0091] MDU-2 Bp: per liter 45 g maltose-1H₂O, 7 g yeast extract, 12 gKH₂PO₄, 1 g MgSO₄-7H₂O, 2 g K₂SO₄, 5 g Urea, 1 g NaCl, 0.5 ml AMG tracemetal solution pH 5.0.

[0092] Materials.

[0093] alpha-³²P-dCTP (3000 Ci/mmol), dNTPs, alpha-³³P-ddNTPs, Hybond-Nmembranes, and DNA labelling beads (-dCTP), T-primed first-strand kit,and Thermo Sequenase kits were from Amersham Pharmacia Biotech (Uppsala,Sweden). TA cloning kits were from Invitrogen (Groningen, TheNetherlands). Taq DNA polymerase and the enhanced avian RT-PCR kit werefrom Sigma (St. Louis, Mo.). Restriction enzymes were from New EnglandBioLabs (Beverly, Mass.). G. graminis was obtained and grown asdescribed by Su and Oliw (supra). Qiagen plant RNeasy mini and OIAquickgel extraction kits were from Merck Eurolab (Stockholm, Sweden).Degenerate primers for PCR were obtained from TIB Molbiol (Berlin,Germany), whereas sequencing primers were purchased from CyberGene(Huddinge, Sweden). 5′-RACE and reverse transcription of total RNA wasperformed with a kit (5′RACE system for rapid amplification of cDNAends) from Life Technologies (Taby, Sweden).

Example 1 Determination of Partial Peptide Sequences of LOX from G.graminis

[0094] A fungal strain of Gaeumannomyces graminis var. tritici wascultivated and lipoxygenase was recovered essentially as described inChao Su and Ernst H. Oliw, J. Biological Chemistry, 273 (21),13072-13079 (1998).

[0095] To obtain data from the N-terminal part of the enzyme,approximately 10 mg of enzyme was analyzed directly by using traditionaledman degradation on the 494 Protein Sequencer, Applied Biosystemsaccording to the manufacturer's instructions.

[0096] Another 40 microgram of sample was lyophilized down to around 20μl and added 20 μl SDS-sample buffer containing DTT before incubation 30min at 37° C. and then boiling the sample for 3 min. 5 t 0.5 Miodoacetamide in 1 M Tris-HCl, pH 7.5 was then added and the sample wasincubated 20 min at room temperature prior to running the sample onSDS-PAGE (4-20%, Novex) according to the manufacturer's instructions.The gel was stained according to standard procedures from Novex.

[0097] The gelpiece (60 kDa) was subsequently cut out and minced with ablade. The gel pieces were washed 2× in 0.5 M tris pH 9.2/ACN (1:1) for45 min at 37° C. The gel pieces were treated with 100% ACN for 10 min tointroduce shrinking of the pieces. The ACN was removed and the piecesdries in speed-Vac. 200 ml 0.1 M NH₄CO₃ (AMBIC) was added and incubatedfor 15 min. AMBIC was removed and 100 ml ACN added. Again incubation for10 min followed by removal of ACN and drying in speed-vac. The cyclewith AMBIC was repeated 2×. After the last drying step 20 ml 0.05 mg/mltrypsin in 0.1 M tris pH 9.2, 10% ACN was added. Incubation for 10 min.Then 300 ml 0.1 M tris pH 9.2, 10% ACN was added. Incubation wascontinued O.N. at 37° C. The supernatant was then removed (saved forcontrol) and the peptides extracted from the gel by adding 30 ml 10%TFA. After 5 min the TFA was withdrawn and collected. Further extractionwas done 2× by adding 150 ml 0.1% TFA, 60% ACN to the gel pieces andincubate for 30 min at 37° C. All extracts were collected (30 ml+150ml+150 ml) and concentrated in the speed-vac to 50 ml. A sample of theconcentrate (5 ml) was run on RP-HPLC on a Vydac C-18 column usingsolvent system of TFA/isopropanol to se if any peptides were present.The rest of the sample was run to collect the peptides. Controls withblank gel pieces were run in parallel. To minimize loss of peptide,selected fractions were sequenced directly without any repurification.

[0098] The resulting N-terminal sequence is shown as SEQ ID NO: 21, andtwo internal peptides (denoted fr 29 and 34) are shown as SEQ ID NOS: 19and 20.

[0099] Further, around 100 μg lipoxygenase was added 40 μl 0.05 Mpotassium phosphate, 10 mM EDTA, 1% Triton X-100, 0.05% SDS, pH 7.3 andheated to 90° C. for 4 min and allowed to cool. Then the sample wasadded 25 mU O-glycosidase (BSA free) and 800 mU EndoF glycosidase(Boehringer) and left over night at 37° C. The sample was then added 75gl SDS sample buffer and run on SDS-PAGE (Novex 4-20%) in 7 lanesaccording to the manufacturer's instructions.

[0100] The 60 kDa bands were cut out from the gel minced and washedtwice in eppendorf tubes with 400 μl of 0.5 M Tris-HCl, pH 9.2:ACN 1:1for 45 min at 37° C. The gel pieces were then treated with 200 μl ACNfor 10 min and then dried in the speed vac. 400 μl NH₄HCO₃ was added andleft for 10 min before removing the supernatant and treating the pieceswith another 200 μl of ACN for 10 min and then drying. 400 μl H₂O wasadded and the sample left for 10 min before repeating the procedure withACN again. The gel pieces was then added 25 μl 0.1 mg/ml trypsin+300 μl0.1 M Tris-HCl, 10% ACN, pH 9.2 and left over night at 37° C. Afterincubation 35 μl of 10 TFA was added and the supernatant were takenafter 30 min for HPLC (Vydac C18, gradient to 80% acetonitril in 0.1%TFA). The gel pieces were then further extracted twice with 150 μl 0.1%TFA, 60% acetonitril. The supernatant was taken and evaporated in thespeed vac to around 50 μl before adding further 100 μl 0.1% TFA and thenre-evaporating down to 50 μl which was then run on the HPLC.

[0101] Three amino acid sequences (denoted fr 20, 21 and 25) wereobtained, as shown in SEQ ID NOS: 16,17 and 18.

Example 2 Cloning of Genomic and cDNA Clone of LOX From G. graminis

[0102] Preparation of Fungal Chromosomal DNA

[0103] A fungal strain Gaeumannomyces graminis var. triftici wascultivated in the YPG (composed per liter: 4 g Yeast extract, 1 gKH₂PO₄, 0.5 g MgSO₄ 7H₂O, 15 g Glucose, pH 6.0) with gentle agitation at25° C. for 6 days. Mycelia was collected by filtration using Mira-cloth(Calbiochem, USA) and washed with deionized water twice. After brieflydried on paper filter, mycelia was frozen by liquid nitrogen and groundby motor on dry ice. Around 0.2 g ground mycelia was put into a 1.5 mleppendorf tube and suspended in 0.5 ml of buffer solution composed with100 mM NaCl, 25 mM EDTA, 1% SDS and 50 mM Tris-HCl (pH 8). Afteraddition of 3 micro-l of 25 mg/ml proteinase K, the tube was incubatedat 65° C. for 30-60 minutes. The solution was extracted with the samevolume of phenol and DNA was precipitated with 0.7 volume of isopropanolat −20° C. The pellet was re-suspended in 0.5 ml of sterilized water andremaining RNA was digested by 50 micro-g of RNase at 37° C. for 30minutes. DNA was phenol extracted and ethanol precipitated again. Thepellet was resuspended in appropriate amount of sterilized water.

[0104] Preparation of mRNA and Synthesis of cDNA

[0105] A fungal strain Gaeumannomyces graminis var. tritici wascultivated in the YPG with gentle agitation at 25° C. for 6 days. Afterthe lipoxygenase activity was confirmed, mycelia was collected andground on dry ice as mentioned before to be used for the preparation oftotal RNA with phenol-chloroform method. Purification of mRNA from totalRNA was performed with Oligo dT cellulose powder (Invitrogen,Netherland).

[0106] Synthesizing of cDNA was done with cDNA Synthesis Kit (Takara,Japan). The first strand cDNA was synthesized using 5-6 micro-g of heatdenatured mRNA as the template in the mixture containing 1.0 mM each ofdNTP, 4 μg of oligo(dT)₁₈ and 2 μg of random primer and 100 U of reversetranscriptase and 1^(st) strand synthesis buffer. In total 50 μl ofreaction mixture was kept at room temperature for 10 min, then incubatedat 422° C. for 1 hour. After the incubation, the reaction mixture waschilled on ice for 2 min and subjected to 2^(nd) strand cDNA synthesis.1138 U of E. coli DNA polymerase and 5 μl of E. coli RNase H/E. coli DNAligase mixture and 2^(nd) DNA synthesis buffer was added to the 1^(st)strand synthesis mixture and diluted up to 240 μl with DEPC-H₂O. Thereaction mixture was incubated at 12° C. 1 hour, 22° C. 1 hour and 70°C. 10 min. Then 10 U of T4 DNA polymerase was added to the reactionmixture and incubated at 37° C. 10 min. Synthesized cDNA was subjectedto agarose gel electrophoresis to confirm the quality.

[0107] Isolation of a Partial Clone of LOX Gene by PCR

[0108] The following primers were designed and synthesized based on theamino acid sequences determined in Example 1. The nucleotide sequence oflinoleate diol synthase of Gaeumannomyces graminis (Genbank Accession #:AF124979) was used as a reference of codon usage.

[0109] Primer 1 for N-term side: SEQ ID NO: 9 (corresponding to aminoacids 1-5 of N-terminal SEQ ID NO: 21).

[0110] Primer 2 for C-term side 1: SEQ ID NO: 10 (corresponding to aminoacids 18-25 of fr 34, SEQ ID NO: 20).

[0111] Primer 3 for C-term side 2: SEQ ID NO: 11 (corresponding to aminoacids 6-15 of fr 34, SEQ ID NO: 20).

[0112] Polymerase chain reaction (PCR) was employed using 0.6 μg ofchromosomal DNA of G. graminis as the template in 50 micro-I reactionmixture containing 2.5 mM each of dNTP, 20 pmol each of primer 1 and 2,2.5 units of LA taq polymerase (Takara, Japan) and GC buffer I suppliedby Takara for LA taq. Reaction condition was shown below. LA taqpolymerase was added to the reaction mixture after step 1. StepTemperature Time 1 98° C. 10 mins 2 96° C. 20 sec 3 53° C. 45 sec 4 72°C. (27 + 3 × cycle) sec 5 72° C. 10 mins

[0113] Second PCR reaction was employed in the reaction mixturedescribed above but using 2 pi of first PCR product as template andprimer 3 instead of primer 2. Reaction condition was the same asdescribed above except step 2 to step 4 were repeated 30 times.

[0114] Amplified 1 kb fragment was gel-purified using QIAquick™ GelExtraction Kit (Qiagen) and subcloned into pT7Blue. Sequence of the PCRclone was determined as shown in SEQ ID NO: 3. From the deduced aminoacid sequence of the PCR fragment, the primer 1 turned out to behybridized to elsewhere than expected, however, amino acid sequence250599Bfr25 (SEQ ID NO: 18) determined in Example 1 was found incontinuous 216 amino acids sequence in the PCR fragment (SEQ ID NO: 8).Identity search showed that the 216 amino acid sequence had the highestidentity to Human 15S Lipoxygenase (Genbank U78294, GENESEOP W93832),Human arachidonate 12-Lipoxygenase (Swiss-Prot P18054) and Plexaurahomomalla 8R-Lipoxygenase (GenBank AF003692, SPTREMBL O16025). Theresults indicated that the obtained PCR fragment contained lipoxygenasegene. The highest score of identity was obtained with Human 15S and wasless than 25%.

[0115] Cloning of Genomic LOX Gene

[0116] To obtain a full-length genomic clone, southern blotting wasemployed on genomic DNA of G. graminis using PCR fragment as a probe.Based on the result, genomic DNA was digested with SalI and separated on1.0% agarose gel. Around 6 kb of DNA digestion was recovered from thegel and ligated with BAP treated pUC19 lineared by Sail. Ligationmixture was transformed into E. coli DH12S to construct a partialgenomic library. It was screened by colony hybridization using the PCRfragment as probe, and a positive E. coli colony was isolated and theplasmid, termed pSG16, was recovered. The plasmid pSG16 contained a 6 kbSalI fragment from G. graminis. Out of 6 kb of this fragment, sequenceof 4.1 kb length including the PCR clone was determined as shown in SEQID NO: 4. The largest open reading frame (ORF) contained theabove-mentioned 216 amino acid sequence as well as the similar sequencesto fr 20, 21, 29 and 34, SEQ ID NOS: 16, 17, 19 and 20 but not theN-terminal sequence (SEQ ID NO: 21) determined in example 1. Two othersmall ORFs were found in the upstream of the largest ORF, but none ofthem had the N-terminal sequence neither. To find the right initial ATGcodon, cDNA cloning was necessary.

[0117] Isolation of cDNA Clone of LOX Gene

[0118] Total RNA was extracted from the mycelia producing lipoxygenaseand subjected for mRNA preparation by Oligo dT cellulose powder. ThecDNA was synthesized from the mRNA using cDNA Synthesis Kit (Takara,Japan) and aiming to obtain full-length cDNA, 1-4 kb of cDNA wasgel-purified to be subjected for the construction of a partial cDNAlibrary. Library was constructed by ligating with the adaptor ofMarathon cDNA Amplification Kit (Clontech, USA), which allows theamplification of aimed cDNA with the Adaptor Primer (AP1) and a customprimer designed for the internal sequence of aimed clone.

[0119] For the amplification of cDNA of LOX, two primers, primer 4 (SEQID NO: 12) and primer 5 (SEQ ID NO: 13), were designed based on thesequence of genomic clone. C-terminal part was amplified with primer 4and AP1, and N-terminal part was amplified with primer 5 and AP1.

[0120] PCR reaction mixture comprised of 2.5 mM dNTP, 30 pmol each ofprimer 4 and AP1 or primer 5 and AP1, 5 units of LA taq polymerase(Takara) and supplied GC buffer 1. Reaction condition was shown below.LA taq polymerase was added to the reaction mixture after step 1. StepTemperature Time 1 98° C. 5 mins 2 95° C. 30 sec 3 74° C. 15 sec 4 68°C. 3 mins 5 95° C. 30 sec 6 95° C. 5 mins 7 54° C. 30 sec 8 68° C. 15sec

[0121] Step 2 to Step 4 were repeated 15 times and the temperature ofStep 3 was decreased 4° C. after each 3 repeat. Step 6 to Step 8 wererepeated 20 times.

[0122] As the results, 0.6 kb and 1.6 kb fragments were amplified for5′-end and 3′-end respectively and the sequences were determined asshown in SEQ ID NO: 5 and SEQ ID NO: 6. Based on the sequence around thepredicted initial ATG and stop codon TAA, the primer 6 (SEQ ID NO: 14)and primer 7 (SEQ ID NO: 15) were designed for the amplification ofend-to-end cDNA. Also desired restriction enzyme sites were introducedat both ends for further plasmid construction.

[0123] Reaction mixture contained 0.08 μg of cDNA library, 2.5 mM dNTP,30 pmol each of primer 6 and 7, 1 units of LA taq polymerase (Takara)and GC buffer. Reaction condition was shown below. LA taq polymerase wasadded to the reaction mixture after step 1. Step Temperature Time 1 98°C. 10 mins 2 96° C. 20 sec 3 53° C. 45 sec 4 72° C. (27 + 3 × cycle) sec5 72° C. 10 mins

[0124] PCR amplified 1.9 kb fragment was isolated and cloned intopT7Blue resulting in pSG26. Sequence of the full-length cDNA wasdetermined. The deduced open reading frame consisted of of 1857 bp,which corresponded to 618 amino acids and a molecular mass of 67600 Da.Comparison with the genomic sequence turned out that the LOX genecontained one intron in the N-terminal side. Predicted N-terminalsequence by signal sequence determination program is “ALPLAAEDAAAT”.Identity search with the full-length amino acid sequence showed that ithad the highest identity to Human 15S Lipoxygenase (Genbank Accessionnumber w93832), less than 25%.

[0125] The plasmid pSG26 was transformed in E. coli JM109 and depositedat DSMZ as DSM 13586 with the accession date Jul. 5, 2000.

Example 3 Expression of G. graminis LOX in A. oryzae

[0126] Host organism

[0127]Aspergillus oryzae BECh2 is described in Danish patent applicationPA 1999 01726. It is a mutant of JaL228 (described in WO98/123000),which is a mutant of IFO4177.

[0128] Transformation of A. oryzae

[0129]Aspergillus oryzae strain BECh2 was inoculated in 100 ml of YPGmedium and incubated at 32° C. for 16 hours with stirring at 80 rpm.Grown mycelia was collected by filtration followed by washing with 0.6 MKCl and re-suspended in 30 ml of 0.6 M KCl containing Glucanex® (NovoNordisk) at the concentration of 30 μl/ml. The mixture was incubated at32° C. with the agitation at 60 rpm until protoplasts were formed. Afterfiltration to remove the remained mycelia, protoplasts were collected bycentrifugation and washed with STC buffer twice. The protoplasts werecounted with a hematitometer and re-suspended in a solution ofSTC:STPC:DMSO (8:2:0.1) to a final concentration of 1.2×10⁷protoplasts/ml. About 4 μg of DNA was added to 100 μl of protoplastsolution, mixed gently and incubated on ice for 30 minutes. 1 μl STPCbuffer was added to the mixture and incubated at 37° C. for another 30minutes. After the addition of 10 ml of Cove top agarose pre-warmed at50° C., the reaction mixture was poured onto COVE-ar agar plates. Theplates were incubated at 32° C. for 5 days.

[0130] SDS-PAGE

[0131] SDS polyacrylamide electrophoresis was carried out using thecommercialized gel PAGEL AE6000 NPU-7.5L (7.5T%) with the apparatusAE-6400 (Atto, Japan) following the provided protocol. 15 μl of samplewas suspended in 15 μl of 2×conc. of sample loading buffer (100 mMTris-HCl (pH 6.8), 200 mM Dithiothreitol, 4% SDS, 0.2% Bromophenol blueand 20% glycerol) and boiled for 5 minutes. 20 μl of sample solution wasapplied to a polyacrylamide gel, and subjected for electrophoresis inthe running buffer (25 mM Tris, 0.1% SDS, 192 mM Glycine) at 20 mA pergel. Resulting gel was stained with Coomassie brilliant blue.

[0132] Construction of Expression Plasmid

[0133] The plasmid pSG26 containing cDNA of G. graminis LOX was digestedby BglII and XhoI and 1.9 kb of fragment which contained the LOX genewas ligated with pMT2188 digested with BamHI and XhoI. The plasmidpMT2188 has a modified Aspergillus niger neutral amylase promoter,Aspergillus nidulans TPI leader sequence, Aspergillus niger glucoamylaseterminator, Aspergillus nidulans amdS gene as a marker for fungaltransformation and S.cerevisiae ura3 as the marker for E.colitransformation. Transformation was done with E. coli DB6507 in whichpyrF gene is deficient and can be complemented with S.cerevisiae Ura3.Resulting plasmid was termed pSG27.

[0134] Expression of G. graminis LOX in A. oryzae

[0135]A. oryzae BECh2 was transformed with the plasmid pSG27 andselection positive transformants were isolated. Transformants were grownon COVE 2-ar at 32° C. for 5 days and inoculated to 100 ml of MS-9shaking flask. After the cultivation with vigorous agitation at 32° C.for 1 day, 3 ml of each culture was transferred to 100 ml of MDU-2 Bp inshaking flask to cultivate at 32° C. for 3 days. Culture broth wascentrifuged at 3500 rpm for 10 minutes and supernatant was collected.Lipoxygenase activities of the supernatant were determinedspectrophotometrically as described before. Positive transformantsshowed about 50,000U/ml culture broth while untransformed A. oryzaeBECh2 showed no activity. Culture supernatant was also subjected toSDS-PAGE analysis. Positive transformants showed 90-110 kDa smear bandwhich indicated the protein was heavily glycosylated. UntransformedA.oryzae BECh2 did not show any major band.

Example 4 Purification of Recombinant Lipoxygenase

[0136] One gram of crude lyophilised lipoxygenase prepared as in theprevious example was dissolved in 40 mL 25 mM Tris-HCl (pH 8.0) and thenfiltered (0.45 μm, type Millex-HV, Millipore). The above and subsequentsteps were all carried out at room temperature. The filtrate was loadedon a SP-Sepharose Fast Flow (2.6×14 cm) with 25 mM Tris-HCl (pH 8.0) at1 mL/min. The column was then washed with the same buffer at 2.5 mL/minuntil baseline was reached (approximately 4 column volumes). The boundprotein was then eluted with a linear gradient from 0 to 330 mM NaCl in25 mM Tris-HCl (pH 8.0) in 2 column volumes. Fractions of 10 mL werecollected. The column was cleaned with 1 M NaCl in 25 mM Tris-HCl (pH8.0). The fractions containing the majority of pure lipoxygenase, asestimated by SDS-PAGE and by activity assay, were pooled andconcentrated using an Amicon cell (10,000 NMWL, YM10, Millipore). Theenzyme was finally transferred into 50 mM sodium phosphate (pH 7.0) bydialysis and stored in aliquots at −20° C. until use.

[0137] SDS-PAGE analysis showed that the lipoxygenase had been purifiedto homogeneity. The enzyme was found to have an estimated molecularweight of 90-110 kDa, somewhat higher than the theoretical value basedon the amino acid sequence (65.6 kDa). This was taken as an indicationof glycosylation. The protein was found to have a very high isoelectricpoint as demonstrated by the successful purification employing cationexchange chromatography.

Example 5 Determination of the Gen and the Deduced Protein Sequ nce ofMn-Lipoxygenase

[0138] 1. Amino Acid Sequences of Internal Peptides and the C-TerminalAmino Acids of Manganese Lipoxygenase

[0139] Manganese lipoxygenase was purified to homogeneity as describedby Su and Oliw (supra), using a strain of G. graminis (different fromthe previous examples). Internal peptides were generated, purified andsequenced by the Sanger method essentially as described for anotherprotein of G. graminis (Hornsten L, Su C, Osbourn A E, Garosi P, HellmanU, Wernstedt C and Oliw E H, Cloning of linoleate diol synthase revealshomology with prostaglandin H synthases. J Biol Chem 274(40): 28219-24,1999). The N-terminal amino acid of Mn-lipoxygenase was blocked, butfour C-terminal amino acid was obtained by C-terminal sequencing.

[0140] (i) C Terminal Amino Acid Sequence

[0141] These C-terminal amino acids were FLSV.

[0142] (ii) Internal Amino Acid Sequences

[0143] The following eight internal amino acid sequences were obtained(where (K), (K/R) and (E) denotes the fact that Lys-C, trypsin and V8cleaves peptides at the C-terminal side of K residues, K or R residues,and E residues, respectively):

[0144] (K)LYTPQPGRYAAACQGLFYLDARSNQFLPLAIK (amino acids 205-237 of SEQID NO: 23 with the substitution K206L)

[0145] (K/R)HPVMGVLNR (amino acids 295-304 of SEQ ID NO: 23 with Lys orArg at position 295)

[0146] (K/R)LFLVDHSYQK (amino acids 196-205 of SEQ ID NO: 23 with Lys orArg at position 196)

[0147] (E)M?AGRGFDGKGLSQG(W/M)PFV (amino acids 569-587 of SEQ ID NO: 23,except that amino acid 570 is uncertain Met and amino acid 584 is Trp orMet)

[0148] (K/R)GLVGEDSGPR (amino acids 365-375 of SEQ ID NO: 23 except thatamino acid 365 was found to be Lys or Arg and 368 Val)

[0149] (K)TNVGADLTYTPLD/AD/WK/LP/ND/NE (amino acids 237-255 of SEQ IDNO: 23 except that amino acid 242 was found to be Ala, 250 Asp or Ala,251 and Asp or Trp)

[0150] (K)G/F SGVLPLHPAw (amino acids 472-483 of SEQ ID NO: 23, exceptthat amino acid 473 was found to be Gly or Phe, and amino acid 483uncertain Trp)

[0151] (K) QTVDDAFAAPDLLAGNGPGRA (amino acids 532-553 of SEQ ID NO: 23except that amino acid 536 was found to be Asp, and 552 Arg)

[0152] 2. RT-PCR with Degenerate Primers Generated cDNA ofMn-Lipoxygenase

[0153] This part of the invention was difficult due to the high GCcontent of the genome of G. graminis.

[0154] Methods for isolation of total RNA from G. graminis andtranscription of mRNA to cDNA had to be optimised. cDNA was oftencontaminated with genomic DNA in spite of digestion with DNAses andother precautions.

[0155] After considerable experimentation, using over 30 degenerateprimers in various combinations, the first cDNA clone of Mn-lipoxygenasecould be obtained by RT-PCR. It was obtained by the following degenerateprimers, which were based on internal peptides 1 and 2 and above. Mn60(5′-AACCAGTTCCTSCCSCTCGCSATCAA) Mn15R (5′-GTCGAGGTAGAAGAGGCCCTGRCAVGC),EO3a (5′-CATCCSGTSATGGGYGTSCTBAA) EOr3a (5′-CGGTTSAGGACRCCCATVACVGGRTG).

[0156] The primers Mn60 and EOr3A generated an RT-PCR band of about230-bp and the primers EO3A and Mn15R generated an RT-PCR band of about220-bp. A sense primer from this sequence (MnS2:5′-CCGTTCAGCGTCGAGAGCAAGG) and an antisense primer from the othersequence (MnS1, 5′-TCTCGGGGATCGTGTGGAAGAGCA) amplified a fragment of337-bp. The amplicon was sequenced and it contained the amino acidsequence of peptide1 in one of the reading frames. The amplicon was usedas probe for Northern blot analysis and for screening of a genomiclibrary (Hornsten et al., supra).

[0157] 3. Screening of a Genomic Library of G. graminis

[0158] A genomic library of G. graminis in Lambda ZAP II was obtained asdescribed by Bowyer P et al., Science 267(5196): 371-4, 1995. It wasscreened with a probe of 0.33-kb from the cDNA sequence. Screening ofover 100 000 plagues yielded 11 positive clones, which were plaguepurified by 2-3 additional rounds of phage screening. The Bluescript SKphagemid was excised with helper phage following published methods.Restriction enzyme analysis showed that all rescued phagemids containedthe same insert of 8-kb.

[0159] 4. Sequencing of the Gene and Coding Region of Mn-LO of G.graminis

[0160] Sequencing was performed of both strands using two differentmethods based on cycle sequencing. The sequencing was difficult due tothe high GC content of the gene (over 60% GC).

[0161] 3.4-kb of the genome of G. graminis was sequenced and thesequence of 2725 nucleotides of the Mn-lipoxygenase gene included anintron of 133-bp. The gene of Mn-lipoxygenase was identified by 5′-RACEfrom the starting point of transcription of 2 mRNA, a¹gcaggttc, and theprotein translation start point A⁷²TG (at nucleotide position 72). TheC-terminal amino acids FLSV were found with the stop codon at position2060-2062. Over 0.6-kb of the 3′-untranslated region was sequenced andtentative polyadenylation signals were found as shown below:

[0162] 5-RACE and cDNA sequencing was used to confirm the deduced openreading frame and the exon-intron borders. The transcription startpoint, the translation start point and the translation end weredetermined as shown in SEQ ID NO: 22 and 23.

[0163] The Intron was found to have a length of 133 bp and to have thesequence shown as SEQ ID NO: 24. It was found to be located betweennucleotides 372 and 373, i.e. between Ser108 and Arg109 of SEQ ID NO:22.

Example 6 Expression of Native and Genetically Modified Mn-Lipoxygenase

[0164] We have subcloned a genomic segment (3-kb) containing the codingregion of the Mn-lipoxygenase gene from the Bluescript SK phagemid intothe multi cloning site (with SpeI and NsiI sites) of the plasmidpGEM-5Zf (Promega) using the restriction enzymes SpeI and NsiI.

[0165] The 5′-end and the intron were modified as follows. pGEM-5Z withthe insert was cleaved with SpeI and BseRI, which cut out the 5′-end ofthe gene and part of the genomic sequence with the intron (1323-bp).This piece was replaced in pGEM with a cDNA sequence of about 405-pb,which was obtained by cleavage of a PCR product of 448-bp with SpeI andBseR1. This vector is designated pGEM_Met. The PCR product was generatedwith a sense primer specific to the translation start region (and withSpeI and NdeI site in the 5′-end of the primer,5′-TTACTAGTCATATGCGCTCCAGGATCCTTGCT), and a gene specific antisenseprimer located at the 3′-end of the BseR1 site. This cDNA part soinserted thus contained the beginning of the ORF (without the Intronpositioned between nucleotides 372 and 373, between Ser108 and Arg109,as shown in the table above), so that the entire ORF was obtained in thevector pGEM_Met.

[0166] The 3′-end was modified with PCR, taking advantage of an BbvCIsite about 130-bp from the stop signal. The sense primer wasgene-specific and located at the 5′-side of the restriction site,whereas the antisense primer was designed from the nucleotides of theterminal amino acids and contained, in addition, NdeI and NsiIrestriction sites. The pGEM_Met vector was cleaved with NsiI and BbvCI,and the excised fragment was replaced with the PCR product cleaved inthe same way. This yielded the vector pGEM-Met_ter. The modified codingregion of Mn-lipoxygenase in this vector can thus be excised with NdeI.All modifications have been confirmed by sequencing of the expressionconstructs.

[0167] 1. Expression of Mn-Lipoxygenase in Procaryotic cells (E. coli)

[0168] The expression vector pET-19b has been linearized with NdeI, andthe modified coding region of Mn-lipoxygenase has been excised with NdeIand ligated into this vector for expression in E coli, as suggested bythe manufacturer of the pET expression vectors (Stratagene). Studies ofrecombinant Mn-lipoxygenase expressed in E. coli is now in progress.

[0169] 2. Expression of Mn-Lipoxygenase in Eukaryotic Cells (Pichiapastoris, Saccharomyces cerevisiae, Aspergillus nidulans, Gaeumannomycesgraminis)

[0170] We plan to use the Pichia Expression kit with the pCIC9 orrelated vectors (Invitrogen), which has to be slightly modified to fitour modified coding region of Mn-lipoxygenase. It is possible thatglycosylation of recombinant Mn-lipoxygenase may differ betweendifferent hosts. We therefore plan to investigate a series of eukaryoticexpression systems in Saccharomyces cerevisiae, Aspergillus nidulans,Gaeumannomyces graminis. Glucosylation may improve the stability of therecombinant enzyme.

[0171] 3. Expression of Mn-Lipoxygenase in Eukaryotic Cells (InsectCells)

[0172] We plan to use the Drosophila Expression System (Schneider 2cells) from Invitrogen using an expression vector without His tags atthe C-terminal end.

[0173] 4. Genetically Modified Mn-Lipoxygenase for Expression.

[0174] Our discovery that Mn-lipoxygenase belongs to the lipoxygenasegene family opens large possibilities for rational modification of thestructure. The 3D sequence of several lipoxygenases are known andMn-lipoxygenase shows significant amino acid identity along manyα-helices of soybean lipoxygenase-1 (Prigge S T, Boyington J C, GaffneyB J and Amzel L M, Structure conservation in lipoxygenases: structuralanalysis of soybean lipoxygenase-1 and modeling of human lipoxygenases.Proteins 24(3): 275-91, 1996), which has been used for modeling of manylipoxygenases. Both the metal ligands and other structurally importantamino acids of Mn-lipoxygenase will be mutated in order to increase thebleaching properties and oxidative properties of the enzyme.

[0175] 4.1 Site Directed Mutagenesis of Amino Acids of ImportantAlpha-Helices.

[0176] Amino acid sequences of Mn-lipoxygenase align with α-helix 9(Prigge et al., supra), which contains the WLLAK sequence and two Hisresidues, which likely are Mn ligands. Systematic changes of amino acidsin this helix might have profound effect on enzyme activity andbleaching properties. In the same way, an amino acid sequence ofMn-Lipoxygenase align with α-helix 18, which contain iron ligands andlikely Mn-ligands (His and Asn). Other predicted α-helices ofMn-lipoxygenase, which should be mutated, correspond to α-helices 7, 8,10-17, 19-22 of soybean lipoxygenase-1 (Prigge et al., supra). Wepredict that some of these genetically modified Mn-lipoxygenases mayhave totally different properties, and the bleaching effect may beenhanced. Predicted Mn ligands thus are 3 His residues, one Asp residueand one Val residue. Mn-lipoxygenase likely belongs to enzymes of the“2-His-1-carboxyl facial triad”.

[0177] 4.2 Site Directed Mutagenesis of Amino Acids of the C-TerminalEnd.

[0178] We plan to mutate the terminal Val to an Ile or to other residuesand to determine the bleaching properties of the mutated form.

[0179] 4.3 Mosaic Forms of Mn-Lipoxygenase

[0180] In order to improve the properties of Mn-lipoxygenase we plansubstitute various parts with the corresponding sequence of soybeanlipoxygenase using the α-helix information described above.

Example 7 Screening of Eukaryotic DNA

[0181] To screen for homologous lipoxygenase genes in eukaryotic fungalstrains, southern hybridization was performed on the genomic DNA fromseveral fungal strains using cDNA of Gaeumannomyces graminis LOX gene asthe probe. Strains of the following species were tested; Pyriculariaoryzae, Psaliota campestris, Penicillium roqueforti and Geotrichumcandidum ATCC34614. Genomic DNA was isolated as described in Example 2.

[0182] The probe was labeled with digoxigenin-dUTP using DIG DNAlabeling Mix (Boehringer Mannheim) as follows; DIG labeled probe wasprepared by PCR using primer 6 (SEQ ID NO: 14) and primer 7 (SEQ ID NO:15) as the full-length cDNA of G. graminis LOX. PCR reaction mixturecontained 0.1 μg of pSG26 as the template, 1.25 mM dNTP, 8% DIG DNALabeling Mix, 30 pmol each of primer 6 and 7, 1 unit of LA taqpolymerase (Takara) and GC buffer. Reaction conditions were as shownbelow. LA taq polymerase was added to the reaction mixture after step 1.Step Temperature Time 1 98° C. 10 mins 2 94° C. 2 mins 3 60° C. 30 sec 472° C. 2 mins 5 72° C. 10 mins

[0183] PCR products were gel-purified and denatured by heating at 98° C.before use.

[0184] About 5 micro-g of DNA digested with restriction enzyme wasseparated on 1.0% agarose gel and denatured by soaking the gel in 0.2NHCl for 30 minutes and in 0.5N NaOH+1.5M NaCl for 30 minutes, then andneutralized in 1M Tris (pH 7.5)+1.5M NaCl for 30 minutes. Denatured DNAwas then transferred to the nylon membrane by vacuum transfer with20×SSC for 15 minutes. After fixing by UV irradiation, nylon membranewas used for the hybridization. Hybridization solution was composed with5×SSC, 0.5% blocking reagent (Boehringer Mannheim), 0.1%N-lauroylsarcosine and 0.02% SDS. The nylon membrane was prehybridizedwith the hybridization solution at 60° C. for 1 hour. After that, theheat-denatured DIG-labeled probe was added to the hybridization solutionand incubated at 60° C. overnight. Resulting membrane was washed withwashing buffer comprising 2×SSC+0.1% SDS for 5 minutes at roomtemperature twice followed by washing with washing buffer 2 composedwith 0.1×SSC+0.1% SDS for 15 minutes at hybridization temperature twice.Washed membrane was air-dried and used for the detection of DIG-labeledDNA by following the provided protocol of DNA detection Kit (BoehringerMannheim).

[0185] As the result, Pyricularia oryzae showed clear positive signalsand Geotrichum candidum showed very weak signals. The results indicatethat Pyricularia oryzae has a lipoxygenase gene that has a high identityto Gaeumannomyces graminis LOX and Geotrichum candidum has a gene thathas low identity to G. graminis LOX.

Example 8 Effect of pH on Mn-Lipoxygenase

[0186] The activity of lipoxygenase produced as in Example 4 was testedat various pH values. The enzyme was found to have a broad pH optimumwith high activity in the range of pH 6-10 or 7-11 with linoleic acid orlinolenic acid as substrate.

[0187] The stability of the enzyme was determined after 1 hourincubation at 40° C. at various pH values. The enzyme was found to havegood stability in the pH range 4-10.

Example 9 Substrate Specificity of Lipoxygenase

[0188] The activity of lipoxygenase produced as in Example 4 was testedon various substrates as described above. The results are expressed ask_(cat) (or V_(max)), K_(M) and k_(cat)/K_(M) according to theMichaelis-Menten equation: k_(cat) K_(M) Substrate micro-mol/min/mg mMk_(cat)/K_(M) Linoleic acid 5.63 0.0068 828 Arachidonic acid 0.2960.0175 16.9 Linoleyl alcohol 3.32 0.0034 982 Methyl linoleate 1.370.164  8.39 Monolinolein 85.4 1,3-dilinolein 12.4 Trilinolein 9.15

[0189] The lipoxygenase showed about twice as high activity towardlinolenic acid than linoleic acid at pH 7.

Example 10 Bleaching of β-Carotene by Native Mn-Lipoxygenas

[0190] Purified Mn-lipoxygenase was used to bleach beta-carotene at pH4.5, 6.5 and 9.5. The highest bleaching activity was found at pH 6.5.

Example 11 Effect of LAS on Mn-Lipoxygenase

[0191] The activity of G. graminis lipoxygenase produced as in Example 4was measured with LAS up to 400 ppm at pH 7.0 and pH 10. Thelipoxygenase was found to be fully stable against LAS up to 400 ppm (thehighest concentration tested) at pH 7 and 10. This indicates that thelipoxygenase is stable enough at normal washing conditions, typically pH10 with 200 ppm of LAS.

1 31 1 1857 DNA Gaeumannomyces graminis CDS (1)..(1854) mat_peptide(49)..() 1 atg cgc tcc agg atc ctt gcc ata gtc ttc gcg gca cgc cat gtggca 48 Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala-15 -10 -5 -1 gcg ctg cca ctc gct gcc gaa gac gct gcg gcg acg ctg tctttg acg 96 Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser LeuThr 1 5 10 15 tcc agc gcc tcc agc acc acc gtg ctc ccg tct ccg acc cagtac acg 144 Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln TyrThr 20 25 30 ctg ccc aac aac gac ccc aac cag ggg gca cgc aac gcc agt atagct 192 Leu Pro Asn Asn Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala35 40 45 cgg aag cgg gag ttg ttc ctc tac ggc cca tcc act ctc ggg cag acg240 Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr 5055 60 acc ttc tac cct acc gga gag ctg ggg aac aac atc tcg gcc cgc gac288 Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp 6570 75 80 gtg cta ctt tgg cgc caa gat gcg gcg aac cag acg gca acg gcg tac336 Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr 8590 95 cgc gaa gcc aat gag acg ttt gca gat att acc agc cgt ggc ggt ttc384 Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe 100105 110 aaa acg ctc gac gac ttt gcg ctc ctc tac aat ggt cac tgg aag gag432 Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu 115120 125 tcg gtt ccg gag ggc ata tcg aag ggc atg ttg agc aac tac acc tcg480 Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser 130135 140 gac ctt ctc ttt tcc atg gag cgg ctg tcc tcc aac cct tac gtc ctc528 Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu 145150 155 160 aag cgc ctc cac cca gcc aag gac aaa ctg ccg ttc agc gtc gagagc 576 Lys Arg Leu His Pro Ala Lys Asp Lys Leu Pro Phe Ser Val Glu Ser165 170 175 aag gtg gtg aag aag ctg acg gcc acc acg ctt gag gcg ctc cacaag 624 Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys180 185 190 ggc ggc cgc ctg ttc ctc gtg gac cac agc tac cag aag aag tacacc 672 Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr195 200 205 ccc cag cca gga cgg tac gcc gcg gcc tgc cag ggg ctt ttc tacctg 720 Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu210 215 220 gac gcg cgg tcc aac caa ttc ctg cct ctg gca atc aag acc aacgtg 768 Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240 ggg gcg gac ctg acg tac acg ccc ctc gac gac aag aac gactgg ctg 816 Gly Ala Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asn Asp TrpLeu 245 250 255 ctg gcc aag atc atg ttc aac aac aac gac ctg ttc tac tcccag atg 864 Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser GlnMet 260 265 270 tac cac gtg ctc ttc cac acg atc ccc gag atc gtg cac gaggcc gcc 912 Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu AlaAla 275 280 285 ttc cgg acg ctg agc gac agg cac ccg gtc atg ggc gtg ctcaac cgc 960 Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu AsnArg 290 295 300 ctc atg tac cag gcc tac gcc atc cgg ccc gtg ggc ggg gctgtg ctc 1008 Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala ValLeu 305 310 315 320 ttc aac ccc ggc ggg ttc tgg gac caa aac ttt ggc ctgccc gcc tcg 1056 Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu ProAla Ser 325 330 335 gcc gcc atc gac ttc ccc ggc tcc gtg tac gcg cag ggcggg ggc ggg 1104 Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly GlyGly Gly 340 345 350 ttc cag gcc ggc tac ctg gag aag gac ctg cgg agc cggggg ctg gtc 1152 Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg GlyLeu Val 355 360 365 ggc gag gac agc ggc ccg cgg ctg ccg cac ttc ccc ttctac gag gac 1200 Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe TyrGlu Asp 370 375 380 gcg cac cgc ctg atc ggg gcg atc cgg cgc ttc atg caggcg ttc gtg 1248 Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln AlaPhe Val 385 390 395 400 gac tcg acg tac ggt gcc gac gac ggc gac gac ggggcg ctg ctg cgc 1296 Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly AlaLeu Leu Arg 405 410 415 gac tac gag ctg cag aac tgg atc gcc gag gcc aacggg ccg gcg cag 1344 Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn GlyPro Ala Gln 420 425 430 gtg cgc gac ttc ccc gcg gcg ccg ctg cgg cgg cgcgca cag ctg gtg 1392 Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg AlaGln Leu Val 435 440 445 gac gtg ctg acg cac gtg gcc tgg gtc acg ggc ggggcg cac cac gtc 1440 Asp Val Leu Thr His Val Ala Trp Val Thr Gly Gly AlaHis His Val 450 455 460 atg aac cag ggc tcg ccc gtc aag ttc tcg ggg gtgctg ccg ctg cac 1488 Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val LeuPro Leu His 465 470 475 480 ccg gcg gcg ctg tac gcg ccc atc ccg acg accaag ggc gcc acc ggc 1536 Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Thr LysGly Ala Thr Gly 485 490 495 aac ggg acg agg gcg ggc ctg ctg gcg tgg ctgccc aac gag cgg cag 1584 Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu ProAsn Glu Arg Gln 500 505 510 gcc gtg gag cag gtc tcg ctg ctc gcg cgc ttcaac cgt gcg cag gtc 1632 Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe AsnArg Ala Gln Val 515 520 525 ggg gac agg aag cag acg gtg cgc gac gcc ttcgcc gcg ccc gac ctg 1680 Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe AlaAla Pro Asp Leu 530 535 540 ctg gcc ggc aac ggg ccg ggg tac gcg gcg gccaac gcg agg ttc gtc 1728 Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala AsnAla Arg Phe Val 545 550 555 560 gag gac acg ggc cgt ata agt cgc gag atggcg ggc aga ggg ttc gac 1776 Glu Asp Thr Gly Arg Ile Ser Arg Glu Met AlaGly Arg Gly Phe Asp 565 570 575 ggc aag ggc ctc agc cag ggc atg ccg ttcgtc tgg acc gcg ctc aat 1824 Gly Lys Gly Leu Ser Gln Gly Met Pro Phe ValTrp Thr Ala Leu Asn 580 585 590 ccc gcc gtc aac cct ttt ttc cta agc gtctaa 1857 Pro Ala Val Asn Pro Phe Phe Leu Ser Val 595 600 2 618 PRTGaeumannomyces graminis 2 Met Arg Ser Arg Ile Leu Ala Ile Val Phe AlaAla Arg His Val Ala -15 -10 -5 -1 Ala Leu Pro Leu Ala Ala Glu Asp AlaAla Ala Thr Leu Ser Leu Thr 1 5 10 15 Ser Ser Ala Ser Ser Thr Thr ValLeu Pro Ser Pro Thr Gln Tyr Thr 20 25 30 Leu Pro Asn Asn Asp Pro Asn GlnGly Ala Arg Asn Ala Ser Ile Ala 35 40 45 Arg Lys Arg Glu Leu Phe Leu TyrGly Pro Ser Thr Leu Gly Gln Thr 50 55 60 Thr Phe Tyr Pro Thr Gly Glu LeuGly Asn Asn Ile Ser Ala Arg Asp 65 70 75 80 Val Leu Leu Trp Arg Gln AspAla Ala Asn Gln Thr Ala Thr Ala Tyr 85 90 95 Arg Glu Ala Asn Glu Thr PheAla Asp Ile Thr Ser Arg Gly Gly Phe 100 105 110 Lys Thr Leu Asp Asp PheAla Leu Leu Tyr Asn Gly His Trp Lys Glu 115 120 125 Ser Val Pro Glu GlyIle Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser 130 135 140 Asp Leu Leu PheSer Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu 145 150 155 160 Lys ArgLeu His Pro Ala Lys Asp Lys Leu Pro Phe Ser Val Glu Ser 165 170 175 LysVal Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys 180 185 190Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr 195 200205 Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu 210215 220 Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240 Gly Ala Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asn AspTrp Leu 245 250 255 Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe TyrSer Gln Met 260 265 270 Tyr His Val Leu Phe His Thr Ile Pro Glu Ile ValHis Glu Ala Ala 275 280 285 Phe Arg Thr Leu Ser Asp Arg His Pro Val MetGly Val Leu Asn Arg 290 295 300 Leu Met Tyr Gln Ala Tyr Ala Ile Arg ProVal Gly Gly Ala Val Leu 305 310 315 320 Phe Asn Pro Gly Gly Phe Trp AspGln Asn Phe Gly Leu Pro Ala Ser 325 330 335 Ala Ala Ile Asp Phe Pro GlySer Val Tyr Ala Gln Gly Gly Gly Gly 340 345 350 Phe Gln Ala Gly Tyr LeuGlu Lys Asp Leu Arg Ser Arg Gly Leu Val 355 360 365 Gly Glu Asp Ser GlyPro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp 370 375 380 Ala His Arg LeuIle Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val 385 390 395 400 Asp SerThr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg 405 410 415 AspTyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln 420 425 430Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val 435 440445 Asp Val Leu Thr His Val Ala Trp Val Thr Gly Gly Ala His His Val 450455 460 Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480 Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Thr Lys Gly AlaThr Gly 485 490 495 Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro AsnGlu Arg Gln 500 505 510 Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe AsnArg Ala Gln Val 515 520 525 Gly Asp Arg Lys Gln Thr Val Arg Asp Ala PheAla Ala Pro Asp Leu 530 535 540 Leu Ala Gly Asn Gly Pro Gly Tyr Ala AlaAla Asn Ala Arg Phe Val 545 550 555 560 Glu Asp Thr Gly Arg Ile Ser ArgGlu Met Ala Gly Arg Gly Phe Asp 565 570 575 Gly Lys Gly Leu Ser Gln GlyMet Pro Phe Val Trp Thr Ala Leu Asn 580 585 590 Pro Ala Val Asn Pro PhePhe Leu Ser Val 595 600 3 1013 DNA Gaeumannomyces graminis 3 gccctgccgaacaacgaccc caaccagggg gcacgcaacg ccagtatagc tcggaagcgg 60 gagttgttcctctacggccc atccactctc gggcagacga ccttctaccc taccggagag 120 ctggggaacaacatctcggc ccgcgacgtg ctactttggc gccaagatgc ggcgaaccag 180 acggcaacggcgtaccgcga agccaatgag acgtttgcag atattaccag cgtatgtgct 240 gatcacatctatgcgtgtag tggccagtct gtttaggagg ctgccagttc ttcctttcgc 300 acttggtattggtacctacc tacccaccta acctaggtac taacacgtct cgttgggcta 360 tagcgtggcggtttcaaaac gctcgacgac tttgcgctcc tctacaatgg tcactggaag 420 gagtcggttccggagggcat atcgaagggc atgttgagca actacacctc ggaccttctc 480 ttttccatggagcggctgtc ctccaaccct tacgtcctca agcgcctcca cccagccaag 540 gacaaactgccgttcagcgt cgagagcaag gtggtgaaga agctgacggc caccacgctt 600 gaggcgctccacaagggcgg ccgcctgttc ctcgtggacc acagctacca gaagaagtgc 660 accccccagccaggacggta cgccgcggcc tgccaggggc ttttctacct ggacgcgcgg 720 tccaaccaattcctgcctct ggcaatcaag accaacgtgg gggcggacct gacgtacacg 780 cccctcgacgacaagaacga ctggctgctg gccaagatca tgttcaacaa caacgacctg 840 ttctactcccagatgtacca cgtgctcttc cacacgatcc ccgagatcgt gcacgaggcc 900 gccttccggacgctgagcga caggcacccg gtcatgggcg tgctcaaccg cctcatgtac 960 caggcctacgccatccggcc cgtgggcggg gccgtgctct tcaaccccgg cgg 1013 4 4098 DNAGaeumannomyces graminis 4 gtcgactcgg cgatgcacgg gccatgtcga attaattcaattccatcgag tcctgcacgc 60 actttaggaa gctccaagcc aaggcactat gaaagttcacaatcgggcat ttgactacca 120 cggcgatttg acgccccagc cgagccgaca ggagcctcaatatcactcat gtgtctgcac 180 atgggcaggc agaccacagc atcccactat ctcttgcgcaccttcttctc acatcagcca 240 aaacactcca ctatcggacc acccgatcag ccctgtacaaatcaaaagaa ccataacaag 300 gtcgctttac caggaatatc cccctcggtg gctgtaagaggttgggtgcc ttgcagagta 360 taagacgttt gtgttcatgt tcctagtctc cctttcctccattcacgctg ccagctgaca 420 ccaagccata tgtctgacta ttcgactgct acactatgcccattgtgata agcccgcgcc 480 gcttaatacc acggaccata catcgaaaac ctcaacttccaagtcggtaa atacgttgtc 540 atgtgatggt agaaggatgc ctcgccgttt ggatcaataaactgtccctt ctgtggtgcg 600 gcccgagacc ccaggattac tcaggctgga taataatatctagctcctcc cccattattt 660 gtgttacttc aaattcgata gatggatggt tcgggcaccctcgtcgctgg aatggcgatc 720 tgcagaaaat ccacacagga ggaacagagc tgacatggaaattgtgaagg agtcggcctg 780 tctgatggcg atggcgaaat tatctcaact agatctctcggtccaacgtc agcctcgtac 840 cagtgatatc gccgtctaca ggtgcctagg aagtactgcgccccgatcat ccgctgtcac 900 agcttcaatg tttcggtctc gccgacatat attgcccatgaaaacgattc aacgtgaggc 960 ggcaacccag tcaagcttcc tattgtcgcc atgaccggtgcaagatgtca ccgcgccggg 1020 cacacgatat ttcttaggca tgccacacac agattgtggcatactagcaa aatctgcctc 1080 tgtttgtgat ccgatggctt gcatcaaaat gcagttcccgtccgtcccgg gctgacagct 1140 ggggtgtcat tggacggatc ggtgcggcca ccacctactaggtgcgatta ttgatactca 1200 acgtgaccaa taagcccagc aatttttccg aacaccctctcgggcatatc caactggagc 1260 taagggggcg gcctgtagga ttcctccgtg acctcatgagagctgagaga gctcagctct 1320 cagctcggtt gagcataagc ccgaagcctt gaccgaggctggaggtgggc gcagtgagac 1380 acccttgagg gccgtgtcct ttagtggcta gaaggatagtgagtatttaa aagtcgagga 1440 aaggctgcat cagcaccatc atgatttccc tttacctctaaggcatttgt gcagtagttc 1500 gctcgttgtt tgcttcttag cccggtagac gctcacgaccaaggctccac cttcgctcga 1560 cgaaatgcgc tccaggatcc ttgccatagt cttcgcggcacgccatgtgg cagcgctgcc 1620 actcgctgcc gaagacgctg cggcgacgct gtctttgacgtccagcgcct ccagcaccac 1680 cgtgctcccg tctccgaccc agtacacgct gcccaacaacgaccccaacc agggggcacg 1740 caacgccagt atagctcgga agcgggagtt gttcctctacggcccatcca ctctcgggca 1800 gacgaccttc taccctaccg gagagctggg gaacaacatctcggcccgcg acgtgctact 1860 ttggcgccaa gatgcggcga accagacggc aacggcgtaccgcgaagcca atgagacgtt 1920 tgcagatatt accagcgtat gtgctgatca catctatgcgtgtagtggcc agtctgttta 1980 ggaggctgcc agttctttct ttcgcacttg gtattggtacctacctaccc acctaaccta 2040 ggtactaaca cgtctcgttg ggctatagcg tggcggtttcaaaacgctcg acgactttgc 2100 gctcctctac aatggtcact ggaaggagtc ggttccggagggcatatcga agggcatgtt 2160 gagcaactac acctcggacc ttctcttttc catggagcggctgtcctcca acccttacgt 2220 cctcaagcgc ctccacccag ccaaggacaa actgccgttcagcgtcgaga gcaaggtggt 2280 gaagaagctg acggccacca cgcttgaggc gctccacaagggcggccgcc tgttcctcgt 2340 ggaccacagc taccagaaga agtacacccc ccagccaggacggtacgccg cggcctgcca 2400 ggggcttttc tacctggacg cgcggtccaa ccaattcctgcctctggcaa tcaagaccaa 2460 cgtgggggcg gacctgacgt acacgcccct cgacgacaagaacgactggc tgctggccaa 2520 gatcatgttc aacaacaacg acctgttcta ctcccagatgtaccacgtgc tcttccacac 2580 gatccccgag atcgtgcacg aggccgcctt ccggacgctgagcgacaggc acccggtcat 2640 gggcgtgctc aaccgcctca tgtaccaggc ctacgccatccggcccgtgg gcggggctgt 2700 gctcttcaac cccggcgggt tctgggacca aaactttggcctgcccgcct cggccgccat 2760 cgacttcccc ggctccgtgt acgcgcaggg cgggggcgggttccaggccg gctacctgga 2820 gaaggacctg cggagccggg ggctggtcgg cgaggacagcggcccgcggc tgccgcactt 2880 ccccttctac gaggacgcgc accgcctgat cggggcgatccggcgcttca tgcaggcgtt 2940 cgtggactcg acgtacggtg ccgacgacgg cgacgacggggcgctgctgc gcgactacga 3000 gctgcagaac tggatcgccg aggccaacgg gccggcgcaggtgcgcgact tccccgcggc 3060 gccgctgcgg cggcgcgcac agctggtgga cgtgctgacgcacgtggcct gggtcacggg 3120 cggggcgcac cacgtcatga accagggctc gcccgtcaagttctcggggg tgctgccgct 3180 gcacccggcg gcgctgtacg cgcccatccc gacgaccaagggcgccaccg gcaacgggac 3240 gagggcgggc ctgctggcgt ggctgcccaa cgagcggcaggccgtggagc aggtctcgct 3300 gctcgcgcgc ttcaaccgtg cgcaggtcgg ggacaggaagcagacggtgc gcgacgcctt 3360 cgccgcgccc gacctgctgg ccggcaacgg gccggggtacgcggcggcca acgcgaggtt 3420 cgtcgaggac acgggccgta taagtcgcga gatggcgggcagagggttcg acggcaaggg 3480 cctcagccag ggcatgccgt tcgtctggac cgcgctcaatcccgccgtca accctttttt 3540 cctaagcgtc taaaaggcct ggccaaagct cagctaattgtggattcggt gtcaaggcct 3600 gtcgccctcg gcgacctgag acgggagatg gggtttatgaagagcgagga tggacattgg 3660 aggtattggg tggtaattaa cagcatgtgg agggagggctacacgagcca aactctgtaa 3720 tggatggcca ccagctgcta gtcagcagtt cccacattccccagaatcac ggctaccgaa 3780 tcgaatgttc acagcacccg actttccatg catatgttcatgtcgccggc ctggttgctt 3840 gcatgcatcc acgtgcgtgc ctggccatgc gagccatgcgagcagtagcc ctggcgacgc 3900 caagggggga caaagcaggc agtgatggag gatggtaacaaccataatgt actttagtct 3960 ggatgcaagt ccgtggctag ggaggaaaaa ggacgtgtctcgcccgcagg aggtagggcg 4020 cggacttttt ggcgaggatg atccaccccc gagcttttccaaatgaagtc atgaccttgg 4080 cataaaatgt gtctcaca 4098 5 575 DNAGaeumannomyces graminis 5 agacgctcac gaccaaggct ccaccttcgc tcgacgaaatgcgctccagg atccttgcca 60 tagtcttcgc ggcacgccat gtggcagcgc tgccactcgctgccgaagac gctgcggcga 120 cgctgtcttt gacgtccagc gcctccagca ccaccgtgctcccgtctccg acccagtaca 180 cgctgcccaa caacgacccc aaccaggggg cacgcaacgccagtatagct cggaagcggg 240 agttgttcct ctacggccca tccactctcg ggcagacgaccttctaccct accggagagc 300 tggggaacaa catctcggcc cgcgacgtgc tactttggcgccaagatgcg gcgaaccaga 360 cggcaacggc gtaccgcgaa gccaatgaga cgtttgcagatattaccagc cgtggcggtt 420 tcaaaacgct cgacgacttt gcgctcctct acaatggtcactggaaggag tcggttccgg 480 agggcatatc gaagggcatg ttgagcaact acacctcggaccttctcttt tccatggagc 540 ggctgtcctc caacccttac gtcctcaagc gcctc 575 61611 DNA Gaeumannomyces graminis 6 cggctgtcct ccaaccctta cgtcctcaagcgcctccacc cagccaagga caaactgccg 60 ttcagcgtcg agagcaaggt ggtgaagaagctgacggcca ccacgcttga ggcgctccac 120 aagggcggcc gcctgttcct cgtggaccacagctaccaga agaagtacac cccccagcca 180 ggacggtacg ccgcggcctg ccaggggcttttctacctgg acgcgcggtc caaccaattc 240 ctgcctctgg caatcaagac caacgtgggggcggacctga cgtacacgcc cctcgacgac 300 aagaacgact ggctgctggc caagatcatgttcaacaaca acgacctgtt ctactcccag 360 atgtaccacg tgctcttcca cacgatccccgagatcgtgc acgaggccgc cttccggacg 420 ctgagcgaca ggcacccggt catgggcgtgctcaaccgcc tcatgtacca ggcctacgcc 480 atccggcccg tgggcggggc tgtgctcttcaaccccggcg ggttctggga ccaaaacttt 540 ggcctgcccg cctcggccgc catcgacttccccggctccg tgtacgcgca gggcgggggc 600 gggttccagg ccggctacct ggagaaggacctgcggagcc gggggctggt cggcgaggac 660 agcggcccgc ggctgccgca cttccccttctacgaggacg cgcaccgcct gatcggggcg 720 atccggcgct tcatgcaggc gttcgtggactcgacgtacg gtgccgacga cggcgacgac 780 ggggcgctgc tgcgcgacta cgagctgcagaactggatcg ccgaggccaa cgggccggcg 840 caggtgcgcg acttccccgc ggcgccgctgcggcggcgcg cacagctggt ggacgtgctg 900 acgcacgtgg cctgggtcac gggcggggcgcaccacgtca tgaaccaggg ctcgcccgtc 960 aagttctcgg gggtgctgcc gctgcacccggcggcgctgt acgcgcccat cccgacgacc 1020 aagggcgcca ccggcaacgg gacgagggcgggcctgctgg cgtggctgcc caacgagcgg 1080 caggccgtgg agcaggtctc gctgctcgcgcgcttcaacc gtgcgcaggt cggggacagg 1140 aagcagacgg tgcgcgacgc cttcgccgcgcccgacctgc tggccggcaa cgggccgggg 1200 tacgcggcgg ccaacgcgag gttcgtcgaggacacgggcc gtataagtcg cgagatggcg 1260 ggcagagggt tcgacggcaa gggcctcagccagggcatgc cgttcgtctg gaccgcgctc 1320 aatcccgccg tcaacccttt tttcctaagcgtctaaaagg cctggccaaa gctcagctaa 1380 ttgtggattc ggtgtcaagg cctgtcgccctcggcgacct gagacgggag atggggttta 1440 tgaagagcga ggatggacat tggaggtattgggtggtaat taacagcatg tggagggagg 1500 gctacacgag ccaaactctg taatggatggccaccagctg ctagtcagca gttcccacat 1560 tccccagaat cacggctacc gaatcgaatgttcacagcaa aaaaaaaaaa a 1611 7 1857 DNA Gaeumannomyces graminis 7atgcgctcca ggatccttgc catagtcttc gcggcacgcc atgtggcagc gctgccactc 60gctgccgaag acgctgcggc gacgctgtct ttgacgtcca gcgcctccag caccaccgtg 120ctcccgtctc cgacccagta cacgctgccc aacaacgacc ccaaccaggg ggcacgcaac 180gccagtatag ctcggaagcg ggagttgttc ctctacggcc catccactct cgggcagacg 240accttctacc ctaccggaga gctggggaac aacatctcgg cccgcgacgt gctactttgg 300cgccaagatg cggcgaacca gacggcaacg gcgtaccgcg aagccaatga gacgtttgca 360gatattacca gccgtggcgg tttcaaaacg ctcgacgact ttgcgctcct ctacaatggt 420cactggaagg agtcggttcc ggagggcata tcgaagggca tgttgagcaa ctacacctcg 480gaccttctct tttccatgga gcggctgtcc tccaaccctt acgtcctcaa gcgcctccac 540ccagccaagg acaaactgcc gttcagcgtc gagagcaagg tggtgaagaa gctgacggcc 600accacgcttg aggcgctcca caagggcggc cgcctgttcc tcgtggacca cagctaccag 660aagaagtaca ccccccagcc aggacggtac gccgcggcct gccaggggct tttctacctg 720gacgcgcggt ccaaccaatt cctgcctctg gcaatcaaga ccaacgtggg ggcggacctg 780acgtacacgc ccctcgacga caagaacgac tggctgctgg ccaagatcat gttcaacaac 840aacgacctgt tctactccca gatgtaccac gtgctcttcc acacgatccc cgagatcgtg 900cacgaggccg ccttccggac gctgagcgac aggcacccgg tcatgggcgt gctcaaccgc 960ctcatgtacc aggcctacgc catccggccc gtgggcgggg ctgtgctctt caaccccggc 1020gggttctggg accaaaactt tggcctgccc gcctcggccg ccatcgactt ccccggctcc 1080gtgtacgcgc agggcggggg cgggttccag gccggctacc tggagaagga cctgcggagc 1140cgggggctgg tcggcgagga cagcggcccg cggctgccgc acttcccctt ctacgaggac 1200gcgcaccgcc tgatcggggc gatccggcgc ttcatgcagg cgttcgtgga ctcgacgtac 1260ggtgccgacg acggcgacga cggggcgctg ctgcgcgact acgagctgca gaactggatc 1320gccgaggcca acgggccggc gcaggtgcgc gacttccccg cggcgccgct gcggcggcgc 1380gcacagctgg tggacgtgct gacgcacgtg gcctgggtca cgggcggggc gcaccacgtc 1440atgaaccagg gctcgcccgt caagttctcg ggggtgctgc cgctgcaccc ggcggcgctg 1500tacgcgccca tcccgacgac caagggcgcc accggcaacg ggacgagggc gggcctgctg 1560gcgtggctgc ccaacgagcg gcaggccgtg gagcaggtct cgctgctcgc gcgcttcaac 1620cgtgcgcagg tcggggacag gaagcagacg gtgcgcgacg ccttcgccgc gcccgacctg 1680ctggccggca acgggccggg gtacgcggcg gccaacgcga ggttcgtcga ggacacgggc 1740cgtataagtc gcgagatggc gggcagaggg ttcgacggca agggcctcag ccagggcatg 1800ccgttcgtct ggaccgcgct caatcccgcc gtcaaccctt ttttcctaag cgtctaa 1857 8216 PRT Gaeumannomyces graminis 8 Arg Gly Gly Phe Lys Thr Leu Asp AspPhe Ala Leu Leu Tyr Asn Gly 1 5 10 15 His Trp Lys Glu Ser Val Pro GluGly Ile Ser Lys Gly Met Leu Ser 20 25 30 Asn Tyr Thr Ser Asp Leu Leu PheSer Met Glu Arg Leu Ser Ser Asn 35 40 45 Pro Tyr Val Leu Lys Arg Leu HisPro Ala Lys Asp Lys Leu Pro Phe 50 55 60 Ser Val Glu Ser Lys Val Val LysLys Leu Thr Ala Thr Thr Leu Glu 65 70 75 80 Ala Leu His Lys Gly Gly ArgLeu Phe Leu Val Asp His Ser Tyr Gln 85 90 95 Lys Lys Cys Thr Pro Gln ProGly Arg Tyr Ala Ala Ala Cys Gln Gly 100 105 110 Leu Phe Tyr Leu Asp AlaArg Ser Asn Gln Phe Leu Pro Leu Ala Ile 115 120 125 Lys Thr Asn Val GlyAla Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys 130 135 140 Asn Asp Trp LeuLeu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe 145 150 155 160 Tyr SerGln Met Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val 165 170 175 HisGlu Ala Ala Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly 180 185 190Val Leu Asn Arg Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly 195 200205 Gly Ala Val Leu Phe Asn Pro Gly 210 215 9 15 DNA Artificial SequencePrimer 9 gccctsccna acaac 15 10 23 DNA Artificial Sequence Primer 10gcsggsaggc cgaagttctg gtc 23 11 29 DNA Artificial Sequence Primer 11ccnccngggt traasagsac sgcsccscc 29 12 36 DNA Artificial Sequence Primer12 cggctgtcct ccaaccctta cgtcctcaag cgcctc 36 13 36 DNA ArtificialSequence Primer 13 gaggcgcttg aggacgtaag ggttggagga cagccg 36 14 36 DNAArtificial Sequence Primer 14 ggaagatcta tgcgctccag gatccttgcc atagtc 3615 38 DNA Artificial Sequence Primer 15 ccgctcgagt tagacgctta ggaaaaaagggttgacgg 38 16 24 PRT Gaeumannomyces graminis 16 Gly Leu Ser Gln Gly MetPro Phe Val Trp Thr Ala Leu Asn Pro Ala 1 5 10 15 Val Asn Pro Phe PheLeu Ser Val 20 17 27 PRT Gaeumannomyces graminis 17 Gly Ala Thr Gly AspGly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro 1 5 10 15 Asp Glu Arg GlnAla Val Glu Gln Val Ser Leu 20 25 18 22 PRT Gaeumannomyces graminis 18Gly Met Leu Ser Asp Tyr Thr Ser Asp Leu Leu Phe Ser Met Glu Arg 1 5 1015 Leu Ser Ser Asn Pro Tyr 20 19 20 PRT Gaeumannomyces graminis 19 PheSer Gly Val Leu Pro Leu His Pro Ala Ala Leu Tyr Ala Pro Ile 1 5 10 15Ile Thr Thr Lys 20 20 25 PRT Gaeumannomyces graminis misc_feature(17)..(17) Unknown 20 Ala Ile Arg Pro Val Gly Gly Ala Val Leu Phe AsnPro Gly Gly Phe 1 5 10 15 Xaa Asp Gln Asn Phe Gly Leu Pro Ala 20 25 2119 PRT Gaeumannomyces graminis misc_feature (6)..(18) Unknown 21 Ala LeuPro Asn Asn Xaa Pro Ala Ala Arg Thr Ala Lys Leu His Xaa 1 5 10 15 LeuXaa Leu 22 1857 DNA Gaeumannomyces graminis CDS (1)..(1854) mat_peptide(49)..() 22 atg cgc tcc agg atc ctt gct ata gtc ttc gca gca cgc cat gtggca 48 Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala-15 -10 -5 -1 gcg ctg cca ctc gct gcc gaa gac gct gcg gcg acg ctg tctttg acg 96 Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser LeuThr 1 5 10 15 tcc agc gcc tcc agc acc acc gtg ctc ccg tct ccg acc cagtac acg 144 Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln TyrThr 20 25 30 ctg ccc aac aaa gac ccc aac cag ggg gca cgc aac gcc agt atagcg 192 Leu Pro Asn Lys Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala35 40 45 cgg aag cgg gag ttg ttc ctc tac ggc cca tcc acg ctc ggg cag acg240 Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr 5055 60 acc ttc tac cct acc gga gag cta ggg aac aat atc tcg gcc cgc gac288 Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp 6570 75 80 gtg ctg ctt tgg cgc caa gat gcg gcg aac cag acg gca acg gcg tac336 Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr 8590 95 cgc gaa gcc aat gag acg ttt gca gat att acc agc cgt ggc ggt ttc384 Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe 100105 110 aaa acg ctc gac gac ttt gcg ctc ctc tac aat ggt cac tgg aag gag432 Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu 115120 125 tcg gtt ccg gag ggc ata tcg aag ggc atg ttg agc aac tac acc tcg480 Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser 130135 140 gac ctt ctc ttt tcc atg gag cgg ctg tcc tcc aac cct tac gtc ctc528 Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu 145150 155 160 aag cgc ctc cac cca acc aag gac aaa ctg ccg ttc agc gtc gagagc 576 Lys Arg Leu His Pro Thr Lys Asp Lys Leu Pro Phe Ser Val Glu Ser165 170 175 aag gtg gtg aag aag ctg acg gcc acc acg ctt gag gcg ctc cacaag 624 Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys180 185 190 ggc ggc cgc ctg ttc ctc gtg gac cac agc tac cag aag aag tacacc 672 Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr195 200 205 ccc cag cca gga cgg tac gcc gcg gcc tgc cag ggg ctt ttc tacctg 720 Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu210 215 220 gac gcg cgg tcc aac cag ttc ctg cct ctg gca atc aag acc aacgtg 768 Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240 ggg gtg gat ctg acg tac acg ccc ctc gac gac aag gac gactgg ctg 816 Gly Val Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asp Asp TrpLeu 245 250 255 ctg gcc aag atc atg ttc aac aac aac gac ctg ttc tac tcccag atg 864 Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser GlnMet 260 265 270 tac cac gtg ctc ttc cac acg atc ccc gag atc gtg cac gaggcc gcc 912 Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu AlaAla 275 280 285 ttc cgg acg ctg agc gac agg cac ccg gtc atg ggc gtg ctcaac cgc 960 Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu AsnArg 290 295 300 ctc atg tac cag gcc tac gcc atc cgg ccc gtg ggc ggg gctgtg ctc 1008 Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala ValLeu 305 310 315 320 ttc aac ccc ggc ggg ttc tgg gac caa aac ttt ggc ctgccc gcc tcg 1056 Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu ProAla Ser 325 330 335 gcc gcc atc gac ttc ccc ggc tcc gtg tac gcg cag ggcggg ggc ggg 1104 Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly GlyGly Gly 340 345 350 ttc cag gcc ggc tac ctg gag aag gac ctg cgg agc cggggg ctg atc 1152 Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg GlyLeu Ile 355 360 365 ggc gag gac agc ggc ccg cgg ctg ccg cac ttc ccc ttctac gag gac 1200 Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe TyrGlu Asp 370 375 380 gcg cac cgc ctg atc ggg gcg atc cgg cgc ttc atg caggcg ttc gtg 1248 Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln AlaPhe Val 385 390 395 400 gac tcg acg tac ggt gcc gac gac ggc gac gac ggggcg ctg ctg cgc 1296 Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly AlaLeu Leu Arg 405 410 415 gac tat gag cta cag aac tgg atc gcc gag gcc aacggg ccg gcg cag 1344 Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn GlyPro Ala Gln 420 425 430 gtg cgc gac ttc ccc gcg gcg ccg ctg cga cgg cgcgcg cag ctg gtg 1392 Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg AlaGln Leu Val 435 440 445 gac gtg ctg acg cac gtg gcc tgg atc acg ggc ggggcg cac cac gtc 1440 Asp Val Leu Thr His Val Ala Trp Ile Thr Gly Gly AlaHis His Val 450 455 460 atg aac cag ggc tcg ccc gtc aag ttc tcg ggg gtgctg ccg ctg cac 1488 Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val LeuPro Leu His 465 470 475 480 ccg gcg gcg ctg tac gcg ccc atc ccg acg gccaag ggc gcc acc ggc 1536 Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Ala LysGly Ala Thr Gly 485 490 495 aac ggg acg agg gcg ggc ctg ctg gcg tgg ctgccc aac gag cgg cag 1584 Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu ProAsn Glu Arg Gln 500 505 510 gcc gtg gag cag gtc tcg ctg ctc gcg cgc ttcaac cgt gcc cag gtc 1632 Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe AsnArg Ala Gln Val 515 520 525 ggg gac agg aag cag acg gtg cgc gac gcc ttcgcc gcg ccc gac ctg 1680 Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe AlaAla Pro Asp Leu 530 535 540 ctg gcc ggc aac ggg ccg ggg tac gcg gcg gccaac gcg agg ttc gtc 1728 Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala AsnAla Arg Phe Val 545 550 555 560 gag gac acg ggc cgt ata agt cgc gag attgcg ggc aga ggg ttt gac 1776 Glu Asp Thr Gly Arg Ile Ser Arg Glu Ile AlaGly Arg Gly Phe Asp 565 570 575 ggc aag ggc ctc agc cag ggc atg ccg ttcgtc tgg acc gcg ctc aat 1824 Gly Lys Gly Leu Ser Gln Gly Met Pro Phe ValTrp Thr Ala Leu Asn 580 585 590 ccc gcc gtc aac cct ttt ttc ctg agc gtctaa 1857 Pro Ala Val Asn Pro Phe Phe Leu Ser Val 595 600 23 618 PRTGaeumannomyces graminis 23 Met Arg Ser Arg Ile Leu Ala Ile Val Phe AlaAla Arg His Val Ala -15 -10 -5 -1 Ala Leu Pro Leu Ala Ala Glu Asp AlaAla Ala Thr Leu Ser Leu Thr 1 5 10 15 Ser Ser Ala Ser Ser Thr Thr ValLeu Pro Ser Pro Thr Gln Tyr Thr 20 25 30 Leu Pro Asn Lys Asp Pro Asn GlnGly Ala Arg Asn Ala Ser Ile Ala 35 40 45 Arg Lys Arg Glu Leu Phe Leu TyrGly Pro Ser Thr Leu Gly Gln Thr 50 55 60 Thr Phe Tyr Pro Thr Gly Glu LeuGly Asn Asn Ile Ser Ala Arg Asp 65 70 75 80 Val Leu Leu Trp Arg Gln AspAla Ala Asn Gln Thr Ala Thr Ala Tyr 85 90 95 Arg Glu Ala Asn Glu Thr PheAla Asp Ile Thr Ser Arg Gly Gly Phe 100 105 110 Lys Thr Leu Asp Asp PheAla Leu Leu Tyr Asn Gly His Trp Lys Glu 115 120 125 Ser Val Pro Glu GlyIle Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser 130 135 140 Asp Leu Leu PheSer Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu 145 150 155 160 Lys ArgLeu His Pro Thr Lys Asp Lys Leu Pro Phe Ser Val Glu Ser 165 170 175 LysVal Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys 180 185 190Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr 195 200205 Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu 210215 220 Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240 Gly Val Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asp AspTrp Leu 245 250 255 Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe TyrSer Gln Met 260 265 270 Tyr His Val Leu Phe His Thr Ile Pro Glu Ile ValHis Glu Ala Ala 275 280 285 Phe Arg Thr Leu Ser Asp Arg His Pro Val MetGly Val Leu Asn Arg 290 295 300 Leu Met Tyr Gln Ala Tyr Ala Ile Arg ProVal Gly Gly Ala Val Leu 305 310 315 320 Phe Asn Pro Gly Gly Phe Trp AspGln Asn Phe Gly Leu Pro Ala Ser 325 330 335 Ala Ala Ile Asp Phe Pro GlySer Val Tyr Ala Gln Gly Gly Gly Gly 340 345 350 Phe Gln Ala Gly Tyr LeuGlu Lys Asp Leu Arg Ser Arg Gly Leu Ile 355 360 365 Gly Glu Asp Ser GlyPro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp 370 375 380 Ala His Arg LeuIle Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val 385 390 395 400 Asp SerThr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg 405 410 415 AspTyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln 420 425 430Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val 435 440445 Asp Val Leu Thr His Val Ala Trp Ile Thr Gly Gly Ala His His Val 450455 460 Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480 Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Ala Lys Gly AlaThr Gly 485 490 495 Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro AsnGlu Arg Gln 500 505 510 Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe AsnArg Ala Gln Val 515 520 525 Gly Asp Arg Lys Gln Thr Val Arg Asp Ala PheAla Ala Pro Asp Leu 530 535 540 Leu Ala Gly Asn Gly Pro Gly Tyr Ala AlaAla Asn Ala Arg Phe Val 545 550 555 560 Glu Asp Thr Gly Arg Ile Ser ArgGlu Ile Ala Gly Arg Gly Phe Asp 565 570 575 Gly Lys Gly Leu Ser Gln GlyMet Pro Phe Val Trp Thr Ala Leu Asn 580 585 590 Pro Ala Val Asn Pro PhePhe Leu Ser Val 595 600 24 133 DNA Gaeumannomyces graminis 24 gtatgtgctgatcacatcta tgcgtgtggt gaccggtctg ctttaggagg ctgccagttc 60 tttctttcgcacttggtatt ggtacctacc tacccaccta acctaggtgc taacacgtct 120 cgttgggctatag 133 25 26 DNA Artificial Sequence Primer 25 aaccagttcc tsccsctcgcsatcaa 26 26 27 DNA Artificial Sequence Primer 26 gtcgaggtag aagaggccctgrcavgc 27 27 23 DNA Artificial Sequence Primer 27 catccsgtsa tgggygtsctbaa 23 28 26 DNA Artificial Sequence Primer 28 cggttsagga crcccatvacvggrtg 26 29 22 DNA Artificial Sequence Primer 29 ccgttcagcg tcgagagcaagg 22 30 24 DNA Artificial Sequence Primer 30 tctcggggat cgtgtggaag agca24 31 32 DNA Artificial Sequence Primer 31 ttactagtca tatgcgctccaggatccttg ct 32

1. A polypeptide having lipoxygenase enzyme activity which: a) has anamino acid sequence which has at least 50% identity with the maturepolypeptide of SEQ ID NO: 2 or 23; b) is encoded by a nucleic acidsequence which hybridizes at 55° C. with a complementary strand of thenucleic acid sequence encoding the mature polypeptide of SEQ ID NO: 1 ora subsequence thereof having at least 100 nucleotides; c) has an aminoacid sequence which can be obtained from the mature polypeptide of SEQID NO: 2 or 23 by substitution, deletion, and/or insertion of one ormore amino acids; or d) is encoded by the lipoxygenase-encoding part ofthe DNA sequence cloned into a plasmid present in Escherichia colideposit number DSM
 13586. 2. A polynucleotide which comprises: a) thepartial DNA sequence encoding a mature lipoxygenase cloned into aplasmid present in Escherichia coli DSM 13586, b) the partial DNAsequence encoding a mature lipoxygenase shown in SEQ ID NO: 2 or 23, c)an analogue of the sequence defined in a) or b) which encodes alipoxygenase and i) has at least 50% identity with said DNA sequence, orii) hybridizes at low stringency with a complementary strand of said DNAsequence or a subsequence thereof having at least 100 nucleotides, iii)is an alletic variant thereof, or d) a complementary strand of a), b) orc).
 3. The polynucleotide of the preceding claim wherein the partial DNAsequence is the mature peptide-coding sequence shown in SEQ ID NO: 1 or22.
 4. A nucleic acid construct comprising the polynucleotide of claim 2or 3 operably linked to one or more control sequences capable ofdirecting the expression of the lipoxygenase in a suitable expressionhost.
 5. A recombinant expression vector comprising the nucleic acidconstruct of claim 4, a promoter, and transcriptional and translationalstop signals.
 6. A recombinant host cell transformed with the nucleicacid construct of claim 4 or the vector of claim
 5. 7. A method forproducing a lipoxygenase comprising a) cultivating the host cell ofclaim 6 under conditions conducive to production of the lipoxygenase,and b) recovering the lipoxygenase.
 8. An oligonucleotide probe whichconsists of at least 20 nucleotides and which encodes a partialpolypeptide sequence of SEQ ID NO: 2 or
 23. 9. A method for obtaining apolypeptide with lipoxygenase activity, comprising: a) preparing aeukaryotic DNA library, b) screening the library to select DNA moleculeswhich hybridize to the probe of claim 8, c) transforming host cells withthe selected DNA molecules, d) cultivating the transformed host cells toexpress polypeptides encoded by the DNA molecules, and e) assaying theexpressed polypeptides to select polypeptides having lipoxygenaseactivity.
 10. A dough composition comprising a manganese lipoxygenase.11. The composition of the preceding claim wherein the lipoxygenase isthe polypeptide of claim
 1. 12. A method for preparing a dough or abaked product made from dough, comprising adding a manganeselipoxygenase to the dough.
 13. A method of oxygenating a substrateselected from the group consisting of linolenic acid, arachidonic acid,linoleyl alcohol and a linoleic acid ester comprising contacting thesubstrate in the presence of oxygen with a manganese lipoxygenase. 14.The method of the preceding claim wherein the ester of linoleic acid ismethyl linoleate, monolinolein, dililnolein or trililnolein.
 15. Themethod of any of claims 12-14 wherein the lipoxygenase is thepolypeptide of claim
 1. 16. A detergent composition comprising amanganese lipoxygenase and a surfactant.
 17. The composition of thepreceding claim wherein the surfactant comprises anionic surfactant,particularly linear alkyl benzenesulfonate.